: The goal of this project is to understand the molecular basis by which mutations alter the activity and inhibition of the HIV-1 Protease and lead to resistance of human immunodeficiency virus against protease inhibitor drugs. The Aims of the project are as follows: Aim 1: to determine the catalytic activities and inhibitor sensitivities of resistant mutants from 14 major mutation positions of HIV-1 PR. The mutations at these 14 positions in the HIV-1 PR polypeptide chain account for the resistance observed in clinical trials of 3 marketed HIV-1 PR inhibitor drugs (Saquanivir, Idinavir, Ritonavir); the mutations also lead to cross-resistance to other major inhibitors under development for clinical use. The kcat/Km and Ki values will be determined for a panel of substrates for each single-residue mutant form of the HIV-1 PR, and for selected multiple-residue mutants. The data will be analyzed quantitatively by a previously established kinetic model. Aim 2: To determine the crystallographic structure of the complexes between inhibitors and resistance mutants of the HIV-1 PR. Crystallographic structures will be determined for chosen single and multiple-site resistant mutants complexed to selected clinically-relevant inhibitors. The differences in the crystal structures between resistant mutants, wild-type, and non-resistant mutants will be critically analyzed and compared to the change in catalytic activities a other kinetic properties. Aim 3: To test hypotheses on the mechanisms of HIV-1 PR catalysis, inhibition, and resistance to inhibitors. Kinetic and structural data will set the basis for testing several hypotheses: (1) Catalytic activities of the resistant mutant PR's determine the order of appearance of the mutants and the steady-state population ratios of the mutants during clinical therapy; (2)A major function of the flaps of the HIV-1 PR is the capturing of the sidechains of substrates and inhibitors at the major specificity positions; (3) Most of the clinically selected resistant mutations of HIV-1 PR worsen the binding of the transition state template (in the enzyme molecule) to the isostere of the inhibitors; (4) Inhibitor drugs bind strongest to HIV-1 PR with a transition state conformation that differs from that of the free enzyme.